Váradi Csaba, Lew Clarence, Guttman András
Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Nagyerdei krt 98, Elméleti tömb, 1/111 H-4032, Debrecen, Hungary.
Anal Chem. 2014 Jun 17;86(12):5682-7. doi: 10.1021/ac501573g. Epub 2014 Jun 9.
Full automation to enable high throughput N-glycosylation profiling and sequencing with good reproducibility is vital to fulfill the contemporary needs of the biopharmaceutical industry and requirements of national regulatory agencies. The most prevalently used glycoanalytical methods of capillary electrophoresis and hydrophilic interaction liquid chromatography, while very efficient, both necessitate extensive sample preparation and cleanup, including glycoprotein capture, N-glycan release, fluorescent derivatization, purification, and preconcentration steps during the process. Currently used protocols to fulfill these tasks require multiple centrifugation and vacuum-centrifugation steps, making liquid handling robot mediated automated sample preparation difficult and expensive. In this paper we report on a rapid magnetic bead based sample preparation approach that enables full automation including all the process phases just in a couple of hours without requiring any centrifugation and/or vacuum centrifugation steps. This novel protocol has been compared to conventional glycan sample preparation strategies using standard glycoproteins (IgG, fetuin, and RNase B) and featured rapid processing time, high release and labeling efficiency, good reproducibility, and the potential of easy automation.
实现全自动化以进行高通量N-糖基化分析和测序并具有良好的重现性，对于满足生物制药行业的当代需求和国家监管机构的要求至关重要。最常用的糖分析方法，如毛细管电泳和亲水相互作用液相色谱，虽然效率很高，但都需要进行大量的样品制备和净化，包括糖蛋白捕获、N-聚糖释放、荧光衍生化、纯化以及过程中的预浓缩步骤。目前用于完成这些任务的方案需要多个离心和真空离心步骤，这使得液体处理机器人介导的自动化样品制备既困难又昂贵。在本文中，我们报告了一种基于磁珠的快速样品制备方法，该方法能够在几个小时内实现包括所有过程阶段的全自动化，而无需任何离心和/或真空离心步骤。该新方案已与使用标准糖蛋白（IgG、胎球蛋白和核糖核酸酶B）的传统聚糖样品制备策略进行了比较，具有处理时间短、释放和标记效率高、重现性好以及易于自动化的潜力。
